Disturbed brain energy metabolism in a rodent model of DYT-TOR1A dystonia.

DYT-TOR1A (DYT1) dystonia, characterized by reduced penetrance and suspected environmental triggers, is explored using a "second hit" DYT-TOR1A rat model. We aim to investigate the biological mechanisms driving the conversion into a dystonic phenotype, focusing on the striatum's role in dystonia pathophysiology. Sciatic nerve crush injury was induced in ∆ETorA rats, lacking spontaneous motor abnormalities, and wild-type (wt) rats. Twelve weeks post-injury, unbiased RNA-sequencing was performed on the striatum to identify differentially expressed genes (DEGs) and pathways. Fenofibrate, a PPARα agonist, was introduced to assess its effects on gene expression. 18F-FDG autoradiography explored metabolic alterations in brain networks. Low transcriptomic variability existed between naïve wt and ∆ETorA rats (17 DEGs). Sciatic nerve injury significantly impacted ∆ETorA rats (1009 DEGs) compared to wt rats (216 DEGs). Pathway analyses revealed disruptions in energy metabolism, specifically in fatty acid ß-oxidation and glucose metabolism. Fenofibrate induced gene expression changes in wt rats but failed in ∆ETorA rats. Fenofibrate increased dystonia-like movements in wt rats but reduced them in ∆ETorA rats. 18F-FDG autoradiography indicated modified glucose metabolism in motor and somatosensory cortices and striatum in both ∆ETorA and wt rats post-injury. Our findings highlight perturbed energy metabolism pathways in DYT-TOR1A dystonia, emphasizing compromised PPARα agonist efficacy in the striatum. Furthermore, we identify impaired glucose metabolism in the brain network, suggesting a potential shift in energy substrate utilization in dystonic DYT-TOR1A rats. These results contribute to understanding the pathophysiology and potential therapeutic targets for DYT-TOR1A dystonia.

Exocyst subunit Exo70B2 is linked to immune signaling and autophagy.

During the immune response, activation of the secretory pathway is key to mounting an effective response, while gauging its output is important to maintain cellular homeostasis. The Exo70 subunit of the exocyst functions as a spatiotemporal regulator by mediating numerous interactions with proteins and lipids. However, a molecular understanding of the exocyst regulation remains challenging. We show that, in Arabidopsis thaliana, Exo70B2 behaves as a bona fide exocyst subunit. Conversely, treatment with the salicylic acid (SA) defence hormone analog benzothiadiazole (BTH), or the immunogenic peptide flg22, induced Exo70B2 transport into the vacuole. We reveal that Exo70B2 interacts with AUTOPHAGY-RELATED PROTEIN 8 (ATG8) via two ATG8-interacting motives (AIMs) and its transport into the vacuole is dependent on autophagy. In line with its role in immunity, we discovered that Exo70B2 interacted with and was phosphorylated by the kinase MPK3. Mimicking phosphorylation had a dual impact on Exo70B2: first, by inhibiting localization at sites of active secretion, and second, it increased the interaction with ATG8. Phosphonull variants displayed higher effector-triggered immunity (ETI) and were hypersensitive to BTH, which induce secretion and autophagy. Our results suggest a molecular mechanism by which phosphorylation diverts Exo70B2 from the secretory into the autophagy pathway for its degradation, to dampen secretory activity.

Does an instructional video as a stand-alone tool promote the acquisition of practical clinical skills? A randomised simulation research trial of skills acquisition and short-term retention.

BACKGROUND: The effectiveness of instructional videos as a stand-alone tool for the acquisition of practical skills is yet unknown because instructional videos are usually didactically embedded. Therefore, we evaluated the acquisition of the skill of a humeral intraosseous access via video in comparison to that of a self-study with an additional retention test. METHODS: After ethical approval, we conducted two consecutive studies. Both were designed as randomised controlled two-armed trials with last-year medical students as independent samples at our institutional simulation centre of a tertiary university hospital centre. In Study 1, we randomly assigned 78 participants to two groups: Vid-Self participants watched an instructional video as an intervention, followed by a test, and after seven days did a self-study as a control, followed by a test. Self-Vid ran through the trial in reverse order. In Study 2, we investigated the influence of the sequence of the two teaching methods on learning success in a new sample of 60 participants: Vid-Self watched an instructional video and directly afterward did the self-study followed by a test, whereas Self-Vid ran through that trial in reverse order. In Studies 1 and 2, the primary outcome was the score (worst score = 0, best score = 20) of the test after intervention and control. The secondary outcome in Study 1 was the change in score after seven days. RESULTS: Study 1: The Vid-Self (Participants n = 42) was superior to the Self-Vid (n = 36) (mean score 14.8 vs. 7.7, p < 0.001). After seven days, Self-vid outperformed Vid-Self (mean score 15.9 vs. 12.5, p < 0.001). Study 2: The Vid-Self (n = 30) and Self-Vid (n = 30) scores did not significantly differ (mean 16.5 vs. mean 16.5, p = 0.97). CONCLUSION: An instructional video as a stand-alone tool effectively promotes the acquisition of practical skills. The best results are yielded by a combination of an instructional video and self-study right after each other, irrespective of sequence. TRIAL REGISTRATIONS: ClinicalTrials.gov: NCT05066204 (13/04/2021) (Study 1) and NCT04842357 (04/10/2021) (Study 2).

Optogenetic control of gene expression in plants in the presence of ambient white light.

Optogenetics is the genetic approach for controlling cellular processes with light. It provides spatiotemporal, quantitative and reversible control over biological signaling and metabolic processes, overcoming limitations of chemically inducible systems. However, optogenetics lags in plant research because ambient light required for growth leads to undesired system activation. We solved this issue by developing plant usable light-switch elements (PULSE), an optogenetic tool for reversibly controlling gene expression in plants under ambient light. PULSE combines a blue-light-regulated repressor with a red-light-inducible switch. Gene expression is only activated under red light and remains inactive under white light or in darkness. Supported by a quantitative mathematical model, we characterized PULSE in protoplasts and achieved high induction rates, and we combined it with CRISPR-Cas9-based technologies to target synthetic signaling and developmental pathways. We applied PULSE to control immune responses in plant leaves and generated Arabidopsis transgenic plants. PULSE opens broad experimental avenues in plant research and biotechnology.

The Medicago truncatula DREPP Protein Triggers Microtubule Fragmentation in Membrane Nanodomains during Symbiotic Infections.

The initiation of intracellular host cell colonization by symbiotic rhizobia in Medicago truncatula requires repolarization of root hairs, including the rearrangement of cytoskeletal filaments. The molecular players governing microtubule (MT) reorganization during rhizobial infections remain to be discovered. Here, we identified M. truncatula DEVELOPMENTALLY REGULATED PLASMA MEMBRANE POLYPEPTIDE (DREPP), a member of the MT binding DREPP/PCaP protein family, and investigated its functions during rhizobial infections. We show that rhizobial colonization of drepp mutant roots as well as transgenic roots overexpressing DREPP is impaired. DREPP relocalizes into symbiosis-specific membrane nanodomains in a stimulus-dependent manner. This subcellular segregation coincides with DREPP-dependent MT fragmentation and a partial loss of the ability to reorganize the MT cytoskeleton in response to rhizobia, which might rely on an interaction between DREPP and the MT-organizing protein SPIRAL2. Taken together, our results reveal that establishment of symbiotic associations in M. truncatula requires DREPP in order to regulate MT reorganization during initial root hair responses to rhizobia.

Establishment of Proximity-Dependent Biotinylation Approaches in Different Plant Model Systems.

Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase or a peroxidase fused to a protein of interest, enabling the covalent biotin labeling of proteins and subsequent capture and identification of interacting and neighboring proteins without the need for the protein complex to remain intact. To date, only a few studies have reported on the use of proximity labeling in plants. Here, we present the results of a systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols that combine mass spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as a test case. We further benchmark the efficiency of various promiscuous biotin ligases in comparison with one-step affinity purification approaches. We identified both known and novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both nonbiotinylated and biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to suggest structural information of protein complexes.

DYT6 mutated THAP1 is a cell type dependent regulator of the SP1 family.

DYT6 dystonia is caused by mutations in the transcription factor THAP1. THAP1 knock-out or knock-in mouse models revealed complex gene expression changes, which are potentially responsible for the pathogenesis of DYT6 dystonia. However, how THAP1 mutations lead to these gene expression alterations and whether the gene expression changes are also reflected in the brain of THAP1 patients are still unclear. In this study we used epigenetic and transcriptomic approaches combined with multiple model systems [THAP1 patients' frontal cortex, THAP1 patients' induced pluripotent stem cell (iPSC)-derived midbrain dopaminergic neurons, THAP1 heterozygous knock-out rat model, and THAP1 heterozygous knock-out SH-SY5Y cell lines] to uncover a novel function of THAP1 and the potential pathogenesis of DYT6 dystonia. We observed that THAP1 targeted only a minority of differentially expressed genes caused by its mutation. THAP1 mutations lead to dysregulation of genes mainly through regulation of SP1 family members, SP1 and SP4, in a cell type dependent manner. Comparing global differentially expressed genes detected in THAP1 patients' iPSC-derived midbrain dopaminergic neurons and THAP1 heterozygous knock-out rat striatum, we observed many common dysregulated genes and 61 of them were involved in dystonic syndrome-related pathways, like synaptic transmission, nervous system development, and locomotor behaviour. Further behavioural and electrophysiological studies confirmed the involvement of these pathways in THAP1 knock-out rats. Taken together, our study characterized the function of THAP1 and contributes to the understanding of the pathogenesis of primary dystonia in humans and rats. As SP1 family members were dysregulated in some neurodegenerative diseases, our data may link THAP1 dystonia to multiple neurological diseases and may thus provide common treatment targets.

NIN-Like Proteins: Interesting Players in Rhizobia-Induced Nitrate Signaling Response During Interaction with Non-Legume Host Arabidopsis thaliana.

Nitrogen is an essential macronutrient and a key cellular messenger. Plants have evolved refined molecular systems to sense the cellular nitrogen status. This is exemplified by the root nodule symbiosis between legumes and symbiotic rhizobia, where nitrate availability inhibits this mutualistic interaction. Additionally, nitrate also functions as a metabolic messenger, resulting in nitrate signaling cascades which intensively crosstalk with other physiological pathways. Nodule inception-like proteins (NLPs) are key players in nitrate signaling and regulate nitrate-dependent transcription during legume-rhizobia interactions. Nevertheless, the coordinated interplay between nitrate signaling pathways and rhizobacteria-induced responses remains to be elucidated. In our study, we investigated rhizobia-induced changes in the root system architecture of the non-legume host arabidopsis under different nitrate conditions. We demonstrate that rhizobium-induced lateral root growth and increased root hair length and density are regulated by a nitrate-related signaling pathway. Key players in this process are AtNLP4 and AtNLP5, because the corresponding mutants failed to respond to rhizobia. At the cellular level, AtNLP4 and AtNLP5 control a rhizobia-induced decrease in cell elongation rates, while additional cell divisions occurred independently of AtNLP4. In summary, our data suggest that root morphological responses to rhizobia are coordinated by a newly considered nitrate-related NLP pathway that is evolutionarily linked to regulatory circuits described in legumes.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Distinct signaling routes mediate intercellular and intracellular rhizobial infection in Lotus japonicus.

Rhizobial infection of legume roots during the development of nitrogen-fixing root nodules can occur intracellularly, through plant-derived infection threads traversing cells, or intercellularly, via bacterial entry between epidermal plant cells. Although it is estimated that around 25% of all legume genera are intercellularly infected, the pathways and mechanisms supporting this process have remained virtually unexplored due to a lack of genetically amenable legumes that exhibit this form of infection. In this study, we report that the model legume Lotus japonicus is infected intercellularly by the IRBG74 strain, recently proposed to belong to the Agrobacterium clade of the Rhizobiaceae. We demonstrate that the resources available for L. japonicus enable insight into the genetic requirements and fine-tuning of the pathway governing intercellular infection in this species. Inoculation of L. japonicus mutants shows that Ethylene-responsive factor required for nodulation 1 (Ern1) and Leu-rich Repeat Receptor-Like Kinase (RinRK1) are dispensable for intercellular infection in contrast to intracellular infection. Other symbiotic genes, including nod factor receptor 5 (NFR5), symbiosis receptor-like kinase (SymRK), Ca2+/calmodulin dependent kinase (CCaMK), exopolysaccharide receptor 3 (Epr3), Cyclops, nodule inception (Nin), nodulation signaling pathway 1 (Nsp1), nodulation signaling pathway 2 (Nsp2), cystathionine-ß-synthase (Cbs), and Vapyrin are equally important for both entry modes. Comparative RNAseq analysis of roots inoculated with IRBG74 revealed a distinctive transcriptome response compared with intracellular colonization. In particular, several cytokinin-related genes were differentially regulated. Corroborating this observation, cyp735A and ipt4 cytokinin biosynthesis mutants were significantly affected in their nodulation with IRBG74, whereas lhk1 cytokinin receptor mutants formed no nodules. These results indicate a differential requirement for cytokinin signaling during intercellular rhizobial entry and highlight distinct modalities of inter- and intracellular infection mechanisms in L. japonicus.

The evolution of dystonia-like movements in TOR1A rats after transient nerve injury is accompanied by dopaminergic dysregulation and abnormal oscillatory activity of a central motor network.

TOR1A is the most common inherited form of dystonia with still unclear pathophysiology and reduced penetrance of 30-40%. ∆ETorA rats mimic the TOR1A disease by expression of the human TOR1A mutation without presenting a dystonic phenotype. We aimed to induce dystonia-like symptoms in male ∆ETorA rats by peripheral nerve injury and to identify central mechanism of dystonia development. Dystonia-like movements (DLM) were assessed using the tail suspension test and implementing a pipeline of deep learning applications. Neuron numbers of striatal parvalbumin+, nNOS+, calretinin+, ChAT+ interneurons and Nissl+ cells were estimated by unbiased stereology. Striatal dopaminergic metabolism was analyzed via in vivo microdialysis, qPCR and western blot. Local field potentials (LFP) were recorded from the central motor network. Deep brain stimulation (DBS) of the entopeduncular nucleus (EP) was performed. Nerve-injured ∆ETorA rats developed long-lasting DLM over 12 weeks. No changes in striatal structure were observed. Dystonic-like ∆ETorA rats presented a higher striatal dopaminergic turnover and stimulus-induced elevation of dopamine efflux compared to the control groups. Higher LFP theta power in the EP of dystonic-like ∆ETorA compared to wt rats was recorded. Chronic EP-DBS over 3 weeks led to improvement of DLM. Our data emphasizes the role of environmental factors in TOR1A symptomatogenesis. LFP analyses indicate that the pathologically enhanced theta power is a physiomarker of DLM. This TOR1A model replicates key features of the human TOR1A pathology on multiple biological levels and is therefore suited for further analysis of dystonia pathomechanism.

The Nanoscale Organization of the Plasma Membrane and Its Importance in Signaling: A Proteolipid Perspective.

Plasma membranes provide a highly selective environment for a large number of transmembrane and membrane-associated proteins. Whereas lateral movement of proteins in this lipid bilayer is possible, it is rather limited in turgid and cell wall-shielded plant cells. However, membrane-resident signaling processes occur on subsecond scales that cannot be explained by simple diffusion models. Accordingly, several receptors and other membrane-associated proteins are organized and functional in membrane nanodomains. Although the general presence of membrane nanodomains has become widely accepted as fact, fundamental functional aspects, the roles of individual lipid species and their interplay with proteins, and aspects of nanodomain maintenance and persistence remain poorly understood. Here, we review the current knowledge of nanodomain organization and function, with a particular focus on signaling processes involving proteins, lipids, and their interactions. Furthermore, we propose new and hypothetical aspects of plant membrane biology that we consider important for future research.

The plasma membrane-associated Ca2+ -binding protein, PCaP1, is required for oligogalacturonide and flagellin-induced priming and immunity.

Early signalling events in response to elicitation include reversible protein phosphorylation and re-localization of plasma membrane (PM) proteins. Oligogalacturonides (OGs) are a class of damage-associated molecular patterns (DAMPs) that act as endogenous signals to activate the plant immune response. Previous data on early phosphoproteome changes in Arabidopsis thaliana upon OG perception uncovered the immune-related phospho-regulation of several membrane proteins, among which PCaP1, a PM-anchored protein with actin filament-severing activity, was chosen for its potential involvement in OG- and flagellin-triggered responses. Here, we demonstrate that PCaP1 is required for late, but not early, responses induced by OGs and flagellin. Moreover, pcap1 mutants, unlike the wild type, are impaired in the recovery of full responsiveness to a second treatment with OGs performed 24 h after the first one. Localization studies on PCaP1 upon OG treatment in plants expressing a functional PCaP1-GFP fusion under the control of PCaP1 promoter revealed fluorescence on the PM, organized in densely packed punctate structures, previously reported as microdomains. Fluorescence was found to be associated also with endocytic vesicles, the number of which rapidly increased after OG treatment, suggesting both an endocytic turnover of PCaP1 for maintaining its homeostasis at the PM and an OG-induced endocytosis.

Symbiotic root infections in Medicago truncatula require remorin-mediated receptor stabilization in membrane nanodomains.

Plant cell infection is tightly controlled by cell surface receptor-like kinases (RLKs). Like other RLKs, the Medicago truncatula entry receptor LYK3 laterally segregates into membrane nanodomains in a stimulus-dependent manner. Although nanodomain localization arises as a generic feature of plant membrane proteins, the molecular mechanisms underlying such dynamic transitions and their functional relevance have remained poorly understood. Here we demonstrate that actin and the flotillin protein FLOT4 form the primary and indispensable core of a specific nanodomain. Infection-dependent induction of the remorin protein and secondary molecular scaffold SYMREM1 results in subsequent recruitment of ligand-activated LYK3 and its stabilization within these membrane subcompartments. Reciprocally, the majority of this LYK3 receptor pool is destabilized at the plasma membrane and undergoes rapid endocytosis in symrem1 mutants on rhizobial inoculation, resulting in premature abortion of host cell infections. These data reveal that receptor recruitment into nanodomains is indispensable for their function during host cell infection.

Impaired dopamine- and adenosine-mediated signaling and plasticity in a novel rodent model for DYT25 dystonia.

Dystonia is a neurological movement disorder characterized by sustained or intermittent involuntary muscle contractions. Loss-of-function mutations in the GNAL gene have been identified to be the cause of "isolated" dystonia DYT25. The GNAL gene encodes for the guanine nucleotide-binding protein G(olf) subunit alpha (Gαolf), which is mainly expressed in the olfactory bulb and the striatum and functions as a modulator during neurotransmission coupling with D1R and A2AR. Previously, heterozygous Gαolf -deficient mice (Gnal+/-) have been generated and showed a mild phenotype at basal condition. In contrast, homozygous deletion of Gnal in mice (Gnal-/-) resulted in a significantly reduced survival rate. In this study, using the CRISPR-Cas9 system we generated and characterized heterozygous Gnal knockout rats (Gnal+/-) with a 13 base pair deletion in the first exon of the rat Gnal splicing variant 2, a major isoform in both human and rat striatum. Gnal+/- rats showed early-onset phenotypes associated with impaired dopamine transmission, including reduction in locomotor activity, deficits in rotarod performance and an abnormal motor skill learning ability. At cellular and molecular level, we found down-regulated Arc expression, increased cell surface distribution of AMPA receptors, and the loss of D2R-dependent corticostriatal long-term depression (LTD) in Gnal+/- rats. Based on the evidence that D2R activity is normally inhibited by adenosine A2ARs, co-localized on the same population of striatal neurons, we show that blockade of A2ARs restores physiological LTD. This animal model may be a valuable tool for investigating Gαolf function and finding a suitable treatment for dystonia associated with deficient dopamine transmission.

Green light for quantitative live-cell imaging in plants.

Plants exhibit an intriguing morphological and physiological plasticity that enables them to thrive in a wide range of environments. To understand the cell biological basis of this unparalleled competence, a number of methodologies have been adapted or developed over the last decades that allow minimal or non-invasive live-cell imaging in the context of tissues. Combined with the ease to generate transgenic reporter lines in specific genetic backgrounds or accessions, we are witnessing a blooming in plant cell biology. However, the imaging of plant cells entails a number of specific challenges, such as high levels of autofluorescence, light scattering that is caused by cell walls and their sensitivity to environmental conditions. Quantitative live-cell imaging in plants therefore requires adapting or developing imaging techniques, as well as mounting and incubation systems, such as micro-fluidics. Here, we discuss some of these obstacles, and review a number of selected state-of-the-art techniques, such as two-photon imaging, light sheet microscopy and variable angle epifluorescence microscopy that allow high performance and minimal invasive live-cell imaging in plants.

Mutant analysis in the nonlegume Parasponia andersonii identifies NIN and NF-YA1 transcription factors as a core genetic network in nitrogen-fixing nodule symbioses.

●Nitrogen-fixing nodulation occurs in 10 taxonomic lineages, with either rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. In the legume-rhizobium endosymbiosis, both processes are guarded by the transcription factor NODULE INCEPTION (NIN) and its downstream target genes of the NUCLEAR FACTOR Y (NF-Y) complex. ●It is hypothesized that nodulation has a single evolutionary origin c. 110 Ma, followed by many independent losses. Despite a significant body of knowledge of the legume-rhizobium symbiosis, it remains elusive which signalling modules are shared between nodulating species in different taxonomic clades. We used Parasponia andersonii to investigate the role of NIN and NF-YA genes in rhizobium nodulation in a nonlegume system. ●Consistent with legumes, P. andersonii PanNIN and PanNF-YA1 are coexpressed in nodules. By analyzing single, double and higher-order CRISPR-Cas9 knockout mutants, we show that nodule organogenesis and early symbiotic expression of PanNF-YA1 are PanNIN-dependent and that PanNF-YA1 is specifically required for intracellular rhizobium infection. ●This demonstrates that NIN and NF-YA1 have conserved symbiotic functions. As Parasponia and legumes diverged soon after the birth of the nodulation trait, we argue that NIN and NF-YA1 represent core transcriptional regulators in this symbiosis.

Flexible fibreoptic intubation in swine - improvement for resident training and animal safety alike.

BACKGROUND: Efficient airway management to facilitate tracheal intubation encompasses essential skills in anaesthesiologic and intensive care. The application of flexible fibreoptic intubation in patients with difficult airways has been identified as the recommended method in various international guidelines. However, providing the opportunity to adequately train residents can be challenging. Using large animals for practice during ongoing studies could help to improve this situation, but there is no recent data on fibreoptic intubation in swine available. METHODS: Thirty male German landrace pigs were anesthetized, instrumented and randomized into two groups. The animals were either intubated conventionally using direct laryngoscopy or a single-use flexible video-endoscope. The intervention was carried out by providers with 3 months experience in conventional intubation of pigs and a brief introduction into endoscopy. Intubation attempts were supervised and aborted, when SpO2 dropped below 93%. After three failed attempts, an experienced supervisor intervened and performed the intubation. Intubation times and attempts were recorded and analysed. RESULTS: Flexible fibreoptic intubation showed a significantly higher success rate in first attempt endotracheal tube placement (75% vs. 47%) with less attempts overall (1.3 ± 0.6 vs. 2.1 ± 1.3, P = 0.043). Conventional intubation was faster (42 s ± 6 s vs. 67 s ± 10s, P < 0.001), but showed a higher complication rate and more desaturation episodes during the trial. CONCLUSIONS: Flexible fibreoptic intubation in swine is feasible and appears to be a safer and more accessible method for inexperienced users to learn. This could not only improve resident training options in hospitals with animal research facilities but might also prevent airway complications and needless animal suffering.

Does the revised intubating laryngeal tube (ILTS-D2) perform better than the intubating laryngeal mask (Fastrach)? - a randomised simulation research study.

BACKGROUND: The intubating laryngeal tube (ILTS-D™) and the intubating laryngeal mask (Fastrach™) are devices that facilitate both extraglottic application and blind tracheal intubation. A revised model of the iLTS-D (for scientific reasons called ILTS-D2) has been designed but not yet evaluated. Therefore, we compared the ILTS-D2 with the established Fastrach under controlled conditions in a prospective randomised controlled simulation research study. METHODS: After ethical approval, we randomised 126 medical students into two groups. Each participant received either Fastrach or ILTS-D2 to perform five consecutive ventilation attempts in a manikin. The primary endpoint was the time to ventilation in the last attempt of using the devices as extraglottic devices. Secondary endpoints were the time to tracheal intubation and the success rates. RESULTS: There was no relevant difference between the two devices in the time to ventilation in the last of five attempts (Fastrach: median 14 s [IQR: 12-15]; ILTS-D2: median 13 s [IQR: 12-15], p = 0.592). Secondary endpoints showed a 2 s faster blind tracheal intubation using the Fastrach than using the ILTS-D2 (Fastrach: median 14 s [IQR: 13-17]; ILTS-D2: median 16 s [IQR: 15-20] p < 0.001). For both devices, the success rates were 100% in the last attempt. CONCLUSIONS: Concerning extraglottic airway management, we could not detect a relevant difference between the revised ILTS-D2 and the Fastrach under laboratory conditions. We advocate for an evaluation of the ILTS-D2 in randomised controlled clinical trials. TRIAL REGISTRATION: Identifier at clinicaltrials.gov: NCT03542747. May 31, 2018.

Mitochondrial defects and neurodegeneration in mice overexpressing wild-type or G399S mutant HtrA2.

The protease HtrA2 has a protective role inside mitochondria, but promotes apoptosis under stress. We previously identified the G399S HtrA2 mutation in Parkinson's disease (PD) patients and reported mitochondrial dysfunction in vitro. Mitochondrial dysfunction is a common feature of PD and related to neurodegeneration. Complete loss of HtrA2 has been shown to cause neurodegeneration in mice. However, the full impact of HtrA2 overexpression or the G399S mutation is still to be determined in vivo. Here, we report the first HtrA2 G399S transgenic mouse model. Our data suggest that the mutation has a dominant-negative effect. We also describe a toxic effect of wild-type (WT) HtrA2 overexpression. Only low overexpression of the G399S mutation allowed viable animals and we suggest that the mutant protein is likely unstable. This is accompanied by reduced mitochondrial respiratory capacity and sensitivity to apoptotic cell death. Mice overexpressing WT HtrA2 were viable, yet these animals have inhibited mitochondrial respiration and significant induction of apoptosis in the brain leading to motor dysfunction, highlighting the opposing roles of HtrA2. Our data further underscore the importance of HtrA2 as a key mediator of mitochondrial function and its fine regulatory role in cell fate. The location and abundance of HtrA2 is tightly controlled and, therefore, human mutations leading to gain- or loss of function could provide significant risk for PD-related neurodegeneration.